Shootin1 Acts in Concert with KIF20B to Promote Polarization of Migrating Neurons

Abstract

Shootin1 has been ascribed a role in regulating polarization of primary hippocampal neurons. To better understand the possible role of Shootin1 in the developing brain, we identified a member of the kinesin superfamily, KIF20B, as a novel Shootin1 interacting protein and a potential mediator of Shootin1 interaction with microtubules. KIF20B/Shootin1 binding was mapped to a 57 aa KIF20B sequence, which was used as a dominant-negative fragment. Direct interaction between that peptide (MBD) and Shootin1 was confirmed by surface plasmon resonance-based technology and the affinity was determined in the 10 Ϫ7 M range. The proteins are expressed in the developing brain and formed a complex in vivo based on coimmunoprecipitation experiments and coimmunostaining in primary neurons. In primary hippocampal neurons Kif20b knockdown reduced Shootin1 mobilization to the developing axon, as evidenced by immunostaining and fluorescence recovery after photobleaching analysis, suggesting that Shootin1 is a novel KIF20B cargo. shRNA targeting of Shootin1 reduced PIP3 accumulation in the growth cone, as did Kif20b shRNA. In the developing mouse brain, Kif20b knockdown or expression of the KIF20B minimal binding domain inhibited neuronal migration, and in vivo migration assays suggested that Shootin1/ Kif20b acts in the same genetic pathway. Time-lapse imaging of multipolar cells in the subventricular zone revealed that downregulating levels of either Shootin1 or Kif20b hindered the transition from multipolar to bipolar cells. Collectively, our data demonstrate the importance of the Shootin1/KIF20B interaction to the dynamic process of pyramidal neuronal polarization and migration.

Microtubules and Growth Cones: Motors Drive the Turn

“…Evidence for such a scenario has been presented here, mainly focusing on three microtubule-based motors (cytoplasmic dynein, kinesin-5 and kinesin-12), and how they contribute to the polarized invasion of microtubules in the growth cone. Various other studies lend support to this motor-based perspective [49,56,57], with some suggesting that other motor proteins also participate [58]. The most exciting aspect of this developing model is regulation, because the activity of each motor is controlled by various factors, such as phosphorylation by particular kinases and/or interaction with co-factors (see Outstanding Questions).…”

The vertebrate-specific Kinesin-6, Kif20b, is required for normal cytokinesis of polarized cortical stem cells and cerebral cortex size

“…The only proteins shown to interact with KIF20B during cytokinesis, PIN1 and PRC1, are upstream regulators (Kamimoto et al, 2001;Kanehira et al, 2007;Lee et al, 2012;Lu and Zhou, 2007). Other candidate cargos of Kif20b (Maliga et al, 2013;Sapir et al, 2013) do not have known roles in cytokinesis.…”

Extracellular and Intracellular Signaling for Neuronal Polarity

“…This transport is important for axon elongation. Kinesin-6 (KIF20B), a mitotic kinesin, interacts with Shootin1 and regulates its transport toward the tips of axons (290). In cooperation with L1 and actin, Shootin1 induces axon specification in cultured neurons and in vivo, generating a driving force in growth cones (307,343,345).…”